Several mouse myeloma proteins important for analysis of in vitro DNA replication have been purified to near homogeneity. These include DNA polymerases alpha and beta and DNA unwinding protein. The native molecular weight of alpha-polymerase is about 300,000, and it contains at least four subunits; the beta-polymerase is one polypeptide chain of 40,000 M.W.; DNA unwinding protein is one polypeptide chain of about 25,000 M.W., however, this protein exhibits microheterogeneity upon isoelectric focusing or high resolution SDS-polyacrylamide gel electrophoresis. Under certain conditions DNA unwinding protein decreases the Tm of poly d(A-T) by 25 degrees. Controlled proteolysis of DNA unwinding protein results in a discrete fragment that is similar to the intact molecule in its avid binding to single-stranded DNA, but different in that it does not decrease the Tm of DNA. Steady state kinetic studies of beta-polymerase have provided data that point to a sequential mechanism for DNA formation. Magnesium activation of this reaction does not change the Km for either dNTP or template-primer, suggesting that Mg2 ion and the substrates bind independently to the enzyme and that Mg2 ion does not seriously affect binding of the substrates.